As influenza is an obligate intracellular pathogen, host cell factors play an essential role in detecting influenza virus, mounting an anti-viral response and modulating viral replication. While reverse genetic approaches have been used to study each of the viral gene products, only recent technological advances have enabled a systematic genetic approach to uncover host factors required in the life cycle of the virus. We have recently developed a genome-wide lentivirus-based RNAi library that can silence human genes in primary and immortalized cell types. We propose to use this RNAi library to identify host factors that are essential for influenza A replication and virus-induced interferon production and to test their value as therapeutic targets in an animal model of influenza infection. We will test the roles of ~10,000 human genes, including: the 'druggable'genome, factors that physically interact with viral proteins that we will identify in a systematic protein interaction screen, genes whose expression is induced or repressed by influenza, other candidate genes and an unbiased collection of genes. To identify essential host factors, A549 lung epithelial cells will be infected with lentiviruses that express shRNAs that individually target each of these human genes. Quantitative measurements of viral and interferon production will be performed to identify bioactive shRNAs that enhance or inhibit these processes. Specific host factors will be selected for further studies if >2 distinct shRNAs targeting a gene silence target gene expression and induce similar phenotypes in primary human lung epithelial cells. The role of selected host factors in each step of the viral life cycle will be determined using assays for viral entry, replication, budding and innate immune signaling. Finally, to assess the prophylactic and therapeutic utility of targeting host factors to counter influenza infection in animals, siRNAs that silence a small number of selected host factors will be administered into mouse lungs. Mice will be infected with influenza before or after siRNA delivery and assessed for viral titer, immune responses, pathology, weight loss and survival. Host factors found to be critical in influenza virus infection in vivo are expected to serve as a new class of targets for small molecule anti-influenza therapeutics that may be less prone to viral resistance. Targeting host factors may thus provide a universal treatment for influenza infection that would address the need for treatment of high-risk groups and preparation for future pandemics.